Tuberculous (TB) meningitis is a serious, life-threatening disease affecting vulnerable populations including HIV-infected individuals and young children. Early diagnosis is challenging and outcomes are poor even with prolonged antimicrobial treatment (?12 months). Although several key antimicrobials have limited central nervous system (CNS) penetration and immunopathology is the major driver of neurological damage, pulmonary TB is still used as the treatment paradigm, with limited efforts to utilize preclinical models of TB meningitis to optimize treatment. We have developed a rabbit model of TB meningitis that replicates key neuropathological features of human disease. Additionally, we have developed several novel, clinically translatable positron emission tomography (PET) tracers to perform holistic, unbiased and noninvasive measurements of pathophysiological processes in live animals. These include 124I-DPA-713 for cerebral inflammation, 11C-rifampin, 76Br-bedaquiline and 18F-linezolid to measure antimicrobial penetration into the CNS, benzothiazinone (BTZ) analogs to directly detect Mycobacterium tuberculosis and 18F-albumin / 11C-verapamil to study blood-brain barrier (BBB) permeability / drug efflux transporter activity respectively. Given the importance of rifampin for the treatment of TB meningitis, we performed detailed pharmacokinetic (PK) studies using dynamic 11C-rifampin PET in rabbits and humans (Tucker et al. Sci Transl Med 2018). We demonstrate that rifampin penetration (area under the curve) into infected-brain lesions is limited, spatially heterogeneous and substantially decreases within two weeks of starting treatment (32% to 11%). Importantly, rifampin concentrations in cerebrospinal fluid (CSF) do not correlate well with those in infected-brain lesions. First-in-human 11C-rifampin PET in a TB meningitis patient was safe, well tolerated and demonstrated similar limited and heterogeneous rifampin penetration. We will utilize these novel imaging tools in the rabbit model to provide mechanistic insights into TB meningitis and key information to optimize treatments: a) measure the penetration of novel TB drugs, including those active against multi-drug resistant TB, into infected-brain lesions as well as provide insights into the relevance of discordant CSF and brain tissue levels; b) compare linezolid and high-dose rifampin based regimens and elucidate the role of BBB permeability and efflux transporters in drug exposures in the CNS; c) perform longitudinal multi-modality imaging to simultaneously visualize intralesional bacterial burden, inflammation and antimicrobial exposure during TB treatments in live animals to correlate the effect of intracerebral inflammatory responses and antimicrobial exposures with the treatment outcome. These assessments are not feasible with current technologies that require resected tissues. This proposal fulfills an important gap in TB drug development and treatment optimization for a devastating disease affecting vulnerable populations.